1. Field of the Invention
The present invention relates to a viral vector construct derived from Epstein-Barr virus (EBV) that targets B-lymphocytes. The viral vector of the invention has a transgene inserted in the EBV major internal repeat region (IR1), for gene delivery and persistent gene expression in B-lymphocytes in vitro and in vivo.
2. Related Art
The terms xe2x80x9cgene transferxe2x80x9d and xe2x80x9cgene therapyxe2x80x9d have been used to describe a variety of methods for delivering genetic material to a cell using viral or non-viral based vector systems. Substantial attention has been given to human gene therapy. The transfer of genetic material to a cell may one day become one of the most important forms of medicine. A variety of public and private institutions now participate in research and development related to the use of genetic material in therapeutic applications. Hundreds of human gene transfer protocols are being conducted at any given time with the approval of the Recombinant DNA Advisory Committee (RAC) and the National Institutes of Health (NIH). Most of these protocols focus on therapy, while others involve marking and non-therapeutic applications. The therapeutic protocols are primarily concerned with infectious diseases, monogenic diseases, and cancer. Gene-based therapies are now expanding into fields such as cardiovascular disease, autoimmune disease, and neurodegenerative disease. The availability of a safe and efficient gene delivery and expression system is essential to the success and efficacy of gene-based therapy.
One method of delivering a gene of interest to a target cell of interest is by using a viral-based vector. Techniques for the formation of vectors or virions are generally described in xe2x80x9cWorking Toward Human Gene Therapy,xe2x80x9d Chapter 28 in Recombinant DNA, 2nd Ed., Watson, J. D. et al., eds., New York: Scientific American Books, pp. 567-581 (1992). An overview of viral vectors or virions that have been used in gene therapy can be found in Wilson, J. M., Clin. Exp. Immunol. 107(Suppl. 1):31-32 (1997), as well as Nakanishi, M., Crit. Rev. Therapeu. Drug Carrier Systems 12:263-310(1995); Robbins, P. D., et al., Trends Biotechnol.16:35-40 (1998); Zhang, J., et al., Cancer Metastasis Rev. 15:385-401 (1996); and Kramm, C. M., et al., Brain Pathology 5:345-381 (1995). Such vectors may be derived from viruses that contain RNA (Vile, R. G., et al., Br. Med Bull. 51:12-30 (1995)) or DNA (Ali M., et al., Gene Ther. 1:367-384 (1994)).
Specific examples of viral vector systems that have been utilized include: retroviruses (Vile, R. G., supra; U.S. Pat. Nos. 5,741,486 and 5,763,242); adenoviruses (Brody, S. L., et al., Ann. N.Y. Acad. Sci. 716: 90-101 (1994); Heise, C. et al., Nat. Med. 3:639-645 (1997)); adenoviral/retroviral chimeras (Bilbao, G., et al., FASEB J. 11:624-634 (1997); Feng, M., et al., Nat. Biotechnol. 15:866-870 (1997)); adeno-associated viruses (Flotte, T. R. and Carter, B. J., Gene Ther. 2:357-362 (1995); U.S. Pat. No. 5,756,283); herpes simplex virus I or II (Latchman, D. S., Mol. Biotechnol. 2:179-195 (1994); U.S. Pat. No. 5,763,217; Chase, M., et al., Nature Biotechnol. 16:444-448 (1998)); parvovirus (Shaughnessy, E., et al., Semin Oncol. 23:159-171 (1996)); reticuloendotheliosis virus (Donburg, R., Gene Therap. 2:301-310 (1995)). Other viruses that can be used as vectors for gene delivery include poliovirus, papillomavirus, vaccinia virus, Epstein-Barr virus (EBV), and lentivirus, as well as hybrid or chimeric vectors incorporating favorable aspects of two or more viruses (Nakanishi, M., Crit. Rev. Therapeu. Drug Carrier Systems 12:263-310 (1995); Zhang, J., et al., Cancer Metastasis Rev. 15:385-401 (1996); Jacoby, D. R., et al., Gene Therapy 4:1281-1283 (1997); Robertson, E. S., et al., Proc.Natl Acad.Sci.USA 93:11334-11340(1996)). Guidance in the construction of gene therapy vectors and the introduction thereof into affected animals for therapeutic purposes may be obtained in the above-referenced publications, as well as U.S. Pat. Nos. 5,631,236, 5,688,773, 5,691,177, 5,670,488, 5,529,774, and 5,601,818.
The viral vectors mentioned above each have advantages and disadvantages. For example, retroviruses have the ability to infect cells and have their genetic material integrated into the host cell with high efficiency. The development of a helper virus free packaging system for retrovirus vectors was a key innovation in the development of this vector system for human gene therapy. Retroviral helper virus free packaging systems generally employ the creation of a stable producer cell line that expresses a selected vector. The relatively small size of the retroviral genome (approximately 11 kb), and the ability to express viral genes without killing cells, allows for the production of a packaging cell line that synthesizes all the proteins required for viral assembly. Producer lines are made by introducing the retroviral vector into such a packaging cell line.
On a down side, however, numerous difficulties with retroviruses have been reported. For example, most retroviral vectors are not capable of gene transfer to postmitotic (nondividing) cells and thus are not applicable to the nervous system because most of the cells in the adult nervous system, especially neurons, are quiescent or postmitotic. In addition, outbreaks of wild-type virus from recombinant virus-producing cell lines have also been reported, with the vector itself causing a disease.
Difficulties have been noted with other viral vectors as well. Adenovirus vectors can only support limited long-term (2 months) gene expression, they appear to be gradually lost from neural cells, and moreover, they can cause both cytopathic effects and an immune response (Le Gal La Salle, G., et al., Science 259:988-990 (1993); Davidson et al., Nat. Genet. 3:219-223 (1993); Yang, Y., et al., J. Virol. 69:2004-2015 (1995)). Adeno-associated virus vectors cause minimal cytopathic effects and can support at least some gene expression for up to 4 months, but gene transfer is inefficient and these vectors can accept only about 4 kb of foreign DNA (Kaplitt, M. G., et al., Nat. Genet. 8:148-154 (1994)).
Vectors based on herpes simplex virus (HSV), and especially HSV-1, have shown promise as potent gene delivery vehicles for several reasons: the virus has a very large genome and thus can accommodate large amounts of foreign DNA (greater than 30 kb), the virus can persist long-term in cells, and can efficiently infect many different cell types, including post-mitotic neural cells (Breakefield, X. O., et al., xe2x80x9cHerpes Simplex Virus Vectors for Tumor Therapy,xe2x80x9d in The Internet Book of Gene Therapy: Cancer Gene Therapeutics, R. E. Sobol and K. J. Scanlon, eds., Appleton and Lange, Stamford, Conn., pp. 41-56 (1995); Glorioso, J. C., et al., xe2x80x9cHerpes Simplex Virus as a Gene-Delivery Vector for the Central Nervous System,xe2x80x9d in Viral Vectors: Gene Therapy and Neuroscience Applications, M. G. Kaplitt and A. D. Loewy, eds., Academic Press, New York, pp. 1-23 (1995)).
Two types of HSV-1 vector systems are known: recombinant and amplicon. Recombinant HSV-1 vectors (Wolfe, J. H. et al., Nat. Genet. 1:379-384 (1992)) are created by inserting genes of interest directly into the 152 kb viral genome, thereby mutating one or more of the approximately 80 viral genes, and concomitantly reducing cytotoxicity. In contrast, HSV-1 amplicons are bacterial plasmids containing only about 1% of the 152 kb HSV-1 genome. They are packaged into HSV-1 particles (virions) using HSV-1 helper virus. HSV-1 amplicons contain: (i) a transgene cassette with a gene of interest; (ii) sequences that allow plasmid propagation in E. coli, such as the origin of DNA replication colE1 and the ampicillin resistance gene; and (iii) non-coding elements of the HSV-1 genome, in particular an origin of DNA replication (ori) and a DNA cleavage/packaging signal (pac), to support replication and subsequent packaging of the amplicon DNA into virions in the presence of helper functions (Spaete, R. R. and Frenkel, N., Cell 30:295-304 (1982)). HSV amplicon vectors are one of the most versatile, efficient, and least toxic, and have the largest transgene capacity of the currently available virus vectors. Because HSV-1 encodes many toxic functions, improvements on the amplicon system have been targeted at reducing the risk associated with the helper virus, or, more recently, using a helper virus-free packaging system (Fraefel, C., et al., J. Virol. 70:7190-7197 (1996); International Patent Publication WO 97/05263, published Feb. 13, 1997); Saeki, Y., et al., Hum Gene Ther 9:2787-2794 (1998)).
In addition to the alpha herpes viruses, such as HSV-1 and HSV-2, described above, viral vectors derived from the gamma herpes virus, Epstein-Barr virus (EBV), have also been used for gene delivery (Robertson, E. S.,et al., Proc.Natl Acad.Sci. USA 93:11334-11340(1996); Sclimenti, C. R., et al., Current Opinion in Biotechnology 9:476-479 (1998); Banerjee, S., et al., Nature Med.1:1303-1308 (December 1995); Cachianes, G., et al., BioTechniques 15:255-259 (1993); Kenney, S.,et al.,Human Gene Therapy 9:1131-1141 (May 1998)). For example, an EBV vector carrying the gene defective in cystic fibrosis provided gene expression for at least 2 months in transformed dividing human airway epithelial cells in culture (Lei, D. C., et al., Gene Therapy 3:427-436 (1996)).
EBV, which is also known as human herpes virus 4, is an about 180-kilobase, double-stranded DNA virus carried in an asymptomatic state by more than 90% of the worldwide adult human population, and is generally maintained as a circular episome in latently infected B cells. It is estimated that one B cell per milliliter of blood from a normal healthy person is infected with EBV.
EBV is highly B-lymphotropic, meaning it mainly infects B lymphocytes, and some epithelial cells. In initiation of latent infection in B lymphocytes, at least 9 viral latent genes are expressed. These viral proteins act in concert to alter B lymphocyte growth and enable the maintenance of the EBV genome as a multicopy episome in a state of latent infection. Each of the viral proteins expressed in latently infected cells, with the exception of the Epstein-Barr nuclear antigen (EBNA1), have epitopes that are presented on the B-cell surface in the context of common class I major histocompatibility complex (MHC) molecules, and are recognized by cytotoxic T lymphocytes. This high level of cytotoxic T-cell recognition and the ability of latently infected cells to shift between full latent gene expression with cell proliferation and an EBNA1-only type of latent infection that is immunologically privileged, enables latently infected cells to achieve a balanced state of long-term persistence in humans. This ability of EBV-infected cells to establish a balanced state of persistence in normal humans raised the possibility that cells infected with EBV recombinants could be used for genetic reconstitution, in vivo (Robertson, E. S. et al., Proc. Natl. Acad. Sci. USA 93:11334-11340 (1996)). The ability of superinfection raised the possibility that cells infected with EBV recombinants could be used in gene therapy for EBV-associated cancers (Paillard, F., Human Gene Therapy 9:1119-1120 (1998)).
Some B-cell lymphomas are EBV positive, especially AIDS-related brain lymphomas, with 100% of malignant cells being EBV positive. Although the role of EBV in the carcinogenesis of lymphomas is still not clear, EBV is certainly not the only factor. Because EBV is able to superinfect EBV-positive B lymphoma cells, an EBV vector carrying a suicide gene could be used to infect and kill the tumor cells. A recombinant EBV vector derived from the P3HR1 strain is an ideal choice because it does not have the EBNA2 gene that is essential for transformation of normal lymphocytes.
There are three difficulties in creating a recombinant EBV as an in vivo gene expression vector. First, the EBV genome is too large to be manipulated as a plasmid for DNA recombination in vitro. Generally, recombinant EBV is generated by homologous recombination. A positive selection marker, such as a gene that encodes an enzyme that inactivates a toxic drug, can be introduced into the EBV genome anywhere. A neomycin resistance gene was introduced into the EBV""s thymidine kinase gene locus (Shimizu, N. et al., J. Virol. 70:7260-7263 (1996)) and a prokaryotic replicon, F factor, was inserted into the BamHI I fragment of the EBV genome (Delecluse, H. -J. et al., Proc. Natl. Acad. Sci. USA 95:8245-8250 (1998)). The later approach allows the generation of viral mutants in E. coli. 
Second, in order to allow for long-term persistence in vivo, the recombinant virus should preserve all the characteristics and functions of the wild-type EBV. That is, the viral vector must be able to complete its full life cycle in EBV""s natural host, B lymphocytes.
Finally, the recombinant virus must be purified and reproduced to a reasonable titer in a cell line that is EBV-negative and supports EBV""s productive cycle. It has been previously reported that EBV-negative Akata lymphoma cells and 293 cells could serve this purpose (Shimizu, N. et al., J. Virol. 70:7260-7263 (1996); Delecluse, H. -J. et al., Proc. Natl. Acad. Sci. USA 95:8245-8250(1998)).
One of the main objectives of gene therapy is to achieve stable, efficient, and persistent genetic modification of target cells. This means that their progeny, or themselves in the case of non-dividing cells, should retain and express the newly introduced genetic material until the end of their lifespan, ideally in a regulated manner. This principle is equally valid when transgenes are introduced to correct genetic deficiencies or for treatment of non-hereditary diseases. The viral vector systems discussed above can achieve retention of the transgene through different mechanisms. For example, retrovirus and AAV vectors can integrate genes into the genome of infected cells, while EBV-derived vectors are maintained by episomal replication.
A comparison of the different viral-based gene delivery systems is summarized below:
Clearly, there is a need in the art for additional and more efficient EBV-based viral vector systems that are capable of stably delivering and persistently expressing a transgene in B-lymphocytes, in vitro or in vivo. Such a vector would be useful for gene therapy of genetic diseases and deficiencies, as well as diseases such as congenital lymphoid immunodeficiencies, such as adenosine deaminase deficiency, hematological disorders, such as hemophilia, B-cell lymphomas, such as AIDS-related brain lymphomas, and other EBV-associated cancers, such as Burkitt""s lymphoma (BL), Hodgkin""s lymphoma, and nasopharyngeal carcinoma.
The purpose of this invention was to find a convenient site in the EBV genome for recombining foreign genes in order to create a non-defective EBV vector that was capable of long-term persistence and expression of foreign genes in B lymphocytes in vivo. This vector would be useful for genetic reconstitution or for the treatment of B-cell lymphomas in humans.
The inventors have surprisingly found that the EBV major internal repeat region (IR1) is an effective site for recombination and expression of foreign genes, without adversely effecting EBV""s latent and lytic life cycles.
The EBV major internal repeat (IR1) region consists of multiple copies of the BamHI xe2x80x9cWxe2x80x9d fragment (3072 bp each), which contains the Wp EBNA promoter and multiple exons for expression of EBNA-LP (Speck,S. H.et al., Proc Natl Acad Sci USA 83:9298-9302 (1986)). The EBNA-LP protein can be expressed from any exon and is essential for latent EBNA expression. The nucleotide sequence of IR1 has been determined (Cheung, A., et al., J. Virol.44:286-294 (1982)).
Since its function was known and the BamHI W copy number can be quite variable, the inventors hypothesized that the EBV IR1 site might be a conducive region for accommodating foreign genes, so long as there was no influence on latent EBNA-LP expression. It was theorized that it would be much easier for a transgene to recombine into one of the multiple targets, than into a single target sequence. Insertion of a foreign gene in the middle area of a 3072 bp W fragment makes it easy for PCR assay to find the targeted recombinant virus.
In order to study the expression of foreign genes from IR1 and the potential effect on EBV-induced B-cell immortalization, latent gene expression, and lytic function, the inventors introduced the enhanced green fluorescent protein (EGFP) gene and a hygromycin phosphotransferase cDNA, controlled by a CMV promoter, into IR1 by homologous recombination in B95-8 cells. Hygromycin-resistant clones were screened by PCR for targeted recombination. The recombinant virus was confirmed by Southern blot analysis. The B95-8 cells containing recombinant virus spontaneously produced both recombinant and wild-type viruses. Pure recombinant virus infected peripheral blood lymphocytes (PBL) or 293 cell clones were selected after infection with the mixed virus preparation from B95-8 cells. Pure recombinant virus preparation can then be obtained from these cells by activation with anti-human IgG, TPA+butyrate or by transfection of BZLF1 gene expressing plasmid. The strategy is shown in FIG. 9.
Southern blot analysis suggested that the insertion of EGFP was in the 7th of 8 BamHI W fragments in IR1. The recombinant EBV, designated EBwgh, was purified in peripheral blood B lymphocytes, 293 cells, and Akata(xe2x88x92) cells. In EBwgh transformed PBLs, Wp transcription and EBNA-LP expression were confirmed by RT-PCR and Western blot analyses, and long-term, high level GFP expression was observed. This indicated that insertion and expression of a foreign gene in any copy of the W fragment has no influence on EBV latent gene expression. EBNA-LP can be expressed directly from downstream sequences of the transgene or spliced over the transgene from the upstream of the transgene, depending on the position of the transgene.
It was concluded that foreign genes can be effectively and persistently expressed from the IR1 region without adverse effect on EBV latent gene expression, and that IR1 is a convenient site for recombining foreign genes into EBV. This non-defective EBV vector thus may be used for gene delivery and persistent expression in B lymphocytes in vivo. The technique to create this EBV vector is simple, easy, and efficient.
Accordingly, the present invention relates to the development and characterization of a gene delivery system based on an EBV vector having a transgene inserted in the EBV IR1 region. In a preferred embodiment, the transgene is inserted in any copy of the EBV IR1 W fragment, provided EBNA-LP expression is not adversely affected.
An exciting property of the vector system of the present invention is the ability of the vector to target B-lymphocytes, and due to a non-integrated episome, to persist in vivo for an extended time, once the vector is introduced. Accordingly, the present invention overcomes the disadvantages of the prior art.
Thus, the present invention provides viral vectors derived from Epstein-Barr Virus (EBV). In the most preferred embodiment, the EBV vector comprises at least one transgene of interest in the EBV major internal repeat region, for gene delivery and persistent expression in B-lymphocytes in vivo.
The transgene(s) may be a reporter or marker gene, and/or a therapeutic gene. Representative examples of suitable reporter genes include: xcex2-galactosidase, green fluorescent protein (gfp), enhanced green fluorescent protein (egfp), galactokinase, alkaline phosphatase, chloramphenicol acetlytransferase, luciferase, and xcex2-lactamase.
Representative examples of suitable selectable marker genes for mammalian cells include genes for blasticidin, histinidol D, hygromycin B, mycophenolic acid, neomycin, puromycin, zeocin, etc.
The therapeutic transgene sequence may be a gene sequence associated with diseases and disorders including, but not limited to genetic diseases and deficiencies, as well as diseases such as congenital lymphoid immunodeficiencies, such as adenosine deaminase deficiency, hematological disorders, such as hemophilia, B-cell lymphomas, such as AIDS-related brain lymphomas, and other EBV-associated cancers, such as Burkitt""s lymphoma (BL), Hodgkin""s lymphoma, and nasopharyngeal carcinoma. Of course, other genetic elements may also be present in the EBV construct, such as additional regulatory, therapeutic, reporter, or marker genes.
The transgene may also be a bacterial artificial chromosome (BAC) backbone for further genetic modification of the EBV viral genome in E. coli. 
The invention further provides a method for expressing a transgene in a B-lymphocyte cell population, in vitro or in vivo using the EBV vectors of the invention. Some exemplary in vivo applications for the gene delivery system of the invention include diseases such as genetic diseases and deficiencies, as well as diseases such as congenital lymphoid immunodeficiencies, such as adenosine deaminase deficiency, hematological disorders, such as hemophilia, B-cell lymphomas, such as AIDS-related brain lymphomas, and other EBV-associated cancers, such as Burkitt""s lymphoma (BL), Hodgkin""s lymphoma, and nasopharyngeal carcinoma.
The invention also provides a method of treating diseases and disorders using the vectors of the invention. Non-limiting examples of the diseases and disorders that can be treated using the present vectors include, for example, the diseases mentioned above.
In another embodiment, the invention provides a method of selectively killing B-lymphocyte neoplasms using the vectors of the invention. The EBV vectors of the invention can be engineered to contain prodrug-activating genes or xe2x80x9csuicide genesxe2x80x9d in the EBV IR1 region.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.